This proposal is divided into two sections. The first section describes experiments to investigate the nature of inverted repeated DNA sequences in mammalian genomes (ir-DNA) and a low molecular weight RNA hydrogen-bonded to poly(A)-terminated heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA), and to determine their positional relationships to messenger RNA coding sequences. The ir-DNA sequences are transcribed into heterogeneous nuclear RNA where they account for approx. 25% of the hnRNA mass; some are also present in polyribosomal, poly(A)-terminated RNA. On the average, each mole of poly(A)-terminated hnRNA contains approx. one mole of the low molecular weight RNA hydrogen-bonded to it. Experiments are also described to investigate the nature of DNA replication origins and to identify them in cloned DNA fragments from the Chinese hamster ovary cell genome. Also described are experiments to ligate the thymidine kinase gene to various DNA fragments of the CHO genome to increase the frequency of transformation of TK- cells to TK+ either by increasing the frequency of integration of TK into the host cell's genome or by replication of the TK gene as a unit independent of the host cell's chromosomes, i.e. as a mini-chromosome. The second section describes experiments to investigate two enzymatic reactions known to play a role in mRMA production and maintenance in eukaryotes, RNA:RNA splicing and end addition to and shortening of poly(A) in mRNA molecules. We have developed an in vitro RNA:RNA splicing reaction that correctly breaks and rejoins RNA molecules to produce mRNAs from their precursor RNA molecules. This system will be purified further to obtain the active components. We have isolated the cytoplasmic poly(A) polymerase from HeLa cells. This enzyme is absolutely dependent on divalent cations, has a mol. wt. of 75,000, and apparently is also a poly(A) endonuclease, leaving a product of approx. 15-18 AMP residues with a 5' pAp and a 3' A-OH. We plan to further purify and characterize these two enzymatic activities as well as determine whether the poly(A)-binding protein, which also has a mol. wt. of 75,000 is in fact the same protein.